• 생약학회지
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• 제39권2호
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• pp.75-79
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• 2008

Nerve growth factor (NGF) is a protein plays a major role in the development and maintenance of central and peripheral nervous system. Recent data suggest that reduced availability of NGF may play a significant role in the pathogenesis of diabetic $polyneuropathy.^{1)}$ In our previous study, steroidal saponin from Liriope platyphylla showed neurotrophic effect by stimulation of NGF synthesis and activation of tyrosine kinase $signaling.^{2)}$ In this study, we examined the neurotrophic effect of Liriope platyphylla (LP); which was from Mylyang(MYL) and Cheongyang(CHE), and Ophiopogon japonicus (CHI) on in vitro and in vivo model for the comparison of their NGF induction. We quantitatively analyzed spicatoside A in the LP and CHI by HPLC. And we investigated the correlation between the contents of spicatoside A and NGF induction efficacy on PC 12 cells and mouse serum. These results suggest that spicatoside A may enhance NGF induction in animal model.

• 생명과학회지
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• 제21권8호
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• pp.1134-1141
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• 2011

TMP21은 AD의 원인으로 작용하는 A${\beta}$-42 펩타이드 생성에 중요한 ${\gamma}$-secretase 활성을 억제하는 p24 family에 속하는 type I 막 단백질이다. 본 연구에서는 TMP21이 세포의 성장과 분화에 중요한 NGF 수용체 신호전달과정에 미치는 영향을 분석하고자 인간의 TMP21 cDNA를 합성하고, CMV promoter 조절 하에 hTMP21를 클로닝하여, CMV/hTMP21 벡터를 제조하였다. 그리고 이들 벡터를 B35 neuroblastoma에서 과발현시킨 후 ${\gamma}$-secretase 구성단백질과 NGF 수용체 연관 단백질의 변화를 관찰하였다. 그 결과, 4종류의 ${\gamma}$-secretase 구성단백질의 발현은 vehicle transfectants보다 CMV/hTMP21 transfectants에서 유의적으로 감소하였다. 또한 NGF low affinity 수용체인 $p75^{NTR}$과 downstream 단백질인 RhoA의 양은 NGF를 처리하지 않은 TMP21 transfectants에서 유의적으로 증가하였으나 NGF 처리에 의해 감소되었다. High affinity NGF 수용체인 TrkA의 인산화도 NGF 처리가 없는 경우 유의적으로 감소하였으나 NGF 처리에 의해 증가되었다. 또한 downstream 신호전달 과정 중에서 ERK의 인산화는 TrkA와 유사한 발현변화를 나타내었으나 Akt 인산화는 NGF의 처리에 의해 더욱 증가하였다. 이러한 결과는 TMP21이 neuroblastoma에서 NGF 수용체 신호전달과정를 조절하는 중요한 단백질로서 작용함을 제시하며, AD의 작용기전 연구에 중요한 기초자료를 제공할 것으로 사료된다.

• Journal of Korean Neurosurgical Society
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• 제59권5호
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• pp.437-441
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• 2016

Objective : Recently, regenerative therapies have been used in clinical trials (heart, cartilage, skeletal). We don't make use of these treatments to spinal cord injury (SCI) patients yet, but regenerative therapies are rising interest in recent study about SCI. Neural precursor/stem cell (NPSC) proliferation is a significant event in functional recovery of the central nervous system (CNS). However, brain NPSCs and spinal cord NPSCs (SC-NPSCs) have many differences including gene expression and proliferation. The purpose of this study was to investigate the influence of neural growth factor (NGF) on the proliferation of SC-NPSCs. Methods : NPSCs ($2{\times}10^4$) were suspended in $100{\mu}L$ of neurobasal medium containing NGF-7S (Sigma-Aldrich) and cultured in a 96-well plate for 12 days. NPSC proliferation was analyzed five times for either concentration of NGF (0.02 and 2 ng/mL). Sixteen rats after SCI were randomly allocated into two groups. In group 1 (SCI-vehicle group, n=8), animals received 1.0 mL of the saline vehicle solution. In group 2 (SCI-NGF group, n=8), the animals received single doses of NGF (Sigma-Aldrich). A dose of 0.02 ng/mL of NGF or normal saline as a vehicle control was intra-thecally injected daily at 24 hour intervals for 7 days. For Immunohistochemistry analysis, rats were sacrificed after one week and the spinal cords were obtained. Results : The elevation of cell proliferation with 0.02 ng/mL NGF was significant (p<0.05) but was not significant for 2 ng/mL NGF. The optical density was increased in the NGF 0.02 ng/mL group compared to the control group and NGF 2 ng/mL groups. The density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group (p<0.05). High power microscopy revealed that the density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group. Conclusion : SC-NPSC proliferation is an important pathway in the functional recovery of SCI. NGF enhances SC-NPSC proliferation in vitro and in vivo. NGF may be a useful option for treatment of SCI patients pending further studies to verify the clinical applicability.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권5호
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제27권5호
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• pp.446-456
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• 2005

Nerve growth factor(NGF) has a critical role in peripheral nerve regeneration. The aim of this study is to construct a well-functioning hNGF-$\beta$ recombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-$\beta$ gene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-$\beta$ which was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-$\beta$ recombinant adenovirus. After production of recombinat adenovirus in a large scale, its transfection efficiency, expression, and function were evaluated using cell lines or primarily cultured cells of HEK293 cells, Schwann cells, fibroblast(NIH3T3) and myocyte(CRH cells). GFP expression was observed in 90% of infected cells compared to uninfected cells. Total mRNA isolated from hNGF-$\beta$ recombinat adenoviru infected cells showed strong RT-PCR band, however, LacZ recombinant adenovirus infected or uninfected cells did not. NGF quantification by ELISA showed a maximal release of 18.865 +/- 0.31ng/mL at 4th day. PC-12 cells exposed to media with hNGF-$\beta$ recombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-$\beta$ recombinant adenovirus and induced over expression of NGF successfully in nonneuronal and neuronal cells. Following these result, it is expected to develop an improved treatment strategy peripheral nerve regeneration using the hNGF-$\beta$ gene transfected cells.

• Animal cells and systems
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• 제1권3호
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• pp.463-466
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• 1997

We have recently demonstrated, by protein and cDNA sequence analysis, that prorenin converting enzyme (PRECE) in the mouse submandibular gland is identical to the epidermal growth factor-binding protein (EGF-BP)type B. However, type A and C did not show prorenin converting activity. To demonstrate whether r-NGF is involved in prorenin processing, we have cloned cDNA of r-NGF and examined prorenin converting activity using the CHO cell expression system, Trypsin converted the 33 kDa r-NGF precursor produced in CHO cells to a two-chain form, 9.4 and 16.4 kDa polypeptide chains, which has been known as an active form of r-NGF in mouse SMG (Server and Shooter, 1976). However, the two chain forms of r-NGF did not reveal prorenin-processing activity. Thus, only PRECE is involved in prorenin processing in mouse SMG. This result shows that their substrate specificities appear to be very strict, although some kallikreins share a high degree of amino acid sequence identity.

• 생물정신의학
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• 제14권3호
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• pp.161-166
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• 2007

Objectives:Recent studies have raised the possibility that nerve growth factor(NGF) is abnormally regulated in the central nervous system(CNS) of animal models with alcohol dependence. The possible alteration of NGF by prolonged alcohol intake may play an important role in alcohol-induced neurotoxicity. Carbohydrate-deficient transferrin(CDT) is regarded as a reliable biological marker of alcohol dependence. The goal of this study was to estimate the changes of %CDT and serum NGF level according to the duration of alcohol abstinence, and to identify whether %CDT level is associated with the serum NGF level in the patients with alcohol dependence. Methods:The subjects were 24 patients with alcohol dependence. We used the Axis-Shield ASA to measure the %CDT level and the enzyme-linked immunosorbent assay(ELISA) to measure the serum NGF level. %CDT and NGF levels were measured immediately after the admission and at 2 weeks after the admission. Results:Decreased %CDT were observed during the period of 2 weeks after the admission. NGF level was not significantly different after 2 weeks. The NGF levels were not correlated with %CDT. The possibility of %CDT as a predictor of alcohol-induced neurotoxicity was not confirmed. Conclusion:Serum NGF levels is not a reliable indicator of abstinence state in the patients with alcohol dependence. Further studies are needed to evaluate the relation between two indicators in regard to hematological and neurological changes in alcohol dependence.

• Molecules and Cells
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• 제21권2호
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• pp.237-243
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• 2006

Brain-derived neurotrophic factor (BDNF) is a neuromodulator of nociceptive responses in the dorsal root ganglia (DRG) and spinal cord. BDNF synthesis increases in response to nerve growth factor (NGF) in trkA-expressing small and medium-sized DRG neurons after inflammation. Previously we demonstrated differential activation of multiple BDNF promoters in the DRG following peripheral nerve injury and inflammation. Using reporter constructs containing individual promoter regions, we investigated the effect of NGF on the multiple BDNF promoters, and the signaling pathway by which NGF activates these promoters in PC12 cells. Although all the promoters were activated 2.4-7.1-fold by NGF treatment, promoter IV gave the greatest induction. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294003, protein kinase A (PKA) inhibitor, H89, and protein kinase C (PKC) inhibitor, chelerythrine, had no effect on activation of promoter IV by NGF. However, activation was completely abolished by the MAPK kinase (MEK) inhibitors, U0126 and PD98059. In addition, these inhibitors blocked NGF-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2. Taken together, these results suggest that the ERK1/2 pathway activates BDNF promoter IV in response to NGF independently of NGF-activated signaling pathways involving PKA and PKC.

• Journal of Korean Neurosurgical Society
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• 제44권6호
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• pp.375-381
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• 2008

Objective : The effects on neural proliferation and differentiation of neural stem cells (NSC) of basic fibroblast growth factor-2 (bFGF). insulin growth factor-I (IGF-I). brain-derived neurotrophic factor (BDNF). and nerve growth factor (NGF) were assessed. Also, following combinations of various factors were investigated : bFGF+IGF-I, bFGF+BDNF, bFGF+NGF, IGF-I+BDNF, IGF-I+NGF, and BDNF+NGF. Methods : Isolated NSC of Fisher 344 rats were cultured with individual growth factors, combinations of factors, and no growth factor (control) for 14 days. A proportion of neurons was analyzed using $\beta$-tubulin III and NeuN as neural markers. Results : Neural differentiations in the presence of individual growth factors for $\beta$-tubulin III-positive cells were : BDNF, 35.3%; IGF-I, 30.9%; bFGF, 18.1%; and NGF, 15.1%, and for NeuN-positive cells was : BDNF, 34.3%; bFGF, 32.2%; IGF-I, 26.6%; and NGF, 24.9%. However, neural differentiations in the absence of growth factor was only 2.6% for $\beta$-tubulin III and 3.1% for NeuN. For $\beta$-tubulin III-positive cells, neural differentiations were evident for the growth factor combinations as follows : bFGF+IGF-I, 73.1 %; bFGF+NGF, 65.4%; bFGF+BDNF, 58.7%; BDNF+IGF-I, 52.2%; NGF+IGF-I, 40.6%; and BDNF+NGF, 40.0%. For NeuN-positive cells : bFGF+IGF-I, 81.9%; bFGF+NGF, 63.5%; bFGF+BDNF, 62.8%; NGF+IGF-I, 62.3%; BDNF+NGF, 56.3%; and BDNF+IGF-I, 46.0%. Significant differences in neural differentiation were evident for single growth factor and combination of growth factors respectively (p<0.05). Conclusion : Combinations of growth factors have an additive effect on neural differentiation. The most prominent neural differentiation results from growth factor combinations involving bFGF and IGF-I. These findings suggest that the combination of a mitogenic action of bFGF and post-mitotic differentiation action of IGF-I synergistically affects neural proliferation and NSC differentiation.

• 생명과학회지
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• 제26권5호
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• pp.509-518
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• 2016

천문동(Asparagus cochinchinensis)은 북아시아 지역에서 열병, 감기, 신장질환, 유방암, 염증질환, 뇌질환 등의 치료에 오랫동안 사용되어온 약용식물(medicinal plant)이다. 비록, 천문동의 항염증(ani-inflammatory) 효능에 대한 일부 연구들이 수행되었지만, 신경세포에서 항염증작용과 신경성장인자(nerve growth factor, NGF)의 연관성에 대한 연구는 수행된바 없다. 따라서, 본 연구에서는 신경세포에서 신경성장인자의 분비와 작용기전에 대한 천문동 열수추출물(aqueous extract from A. cochinchinensis, AEAC)의 영향을 연구하였다. AEAC로 처리된 B35세포의 배양액에 NGF단백질의 농도는 대조물질(vehicle) 처리군에 비하여 유의적으로 증가하였으며, 특별한 독성은 관찰되지 않았다. 또한, NGF mRNA의 발현도 단백질의 농도변화와 유사한 양상을 나타내었다. 더불어, B35세포로부터 분비된 NGF의 생리활성을 확인하기 위해, AEAC-조정배지(conditioned medium)를 미분화된 PC12세포에 처리한 후 이들 세포의 신경염성 성장(neuritic outgrowth)을 관찰하였다. PC12세포의 수상돌기 길이(dendritic length)는 vehicle처리군에 비하여 AEAC-조정배지처리군에서 유의적으로 증가하였다. 또한, High affinity NGF 수용체의 하위신호전달에 포함된 p-TrkA와 p-ERK의 발현은 AEAC-조정배지처리군에서 높았지만, low affinity NGF 수용체의 하위신호전달에서는 낮은 수준으로 관찰되었다. 이러한 결과는 AEAC가 신경세포에서 NGF발현과 분비의 조절에 기여하기 때문에 신경퇴행성질환(neurodegenerative disease) 치료제로서 우수한 후보물질임을 제시하고 있다.